EP0054676A2 - Process for determining HL antigens - Google Patents

Process for determining HL antigens Download PDF

Info

Publication number
EP0054676A2
EP0054676A2 EP81108755A EP81108755A EP0054676A2 EP 0054676 A2 EP0054676 A2 EP 0054676A2 EP 81108755 A EP81108755 A EP 81108755A EP 81108755 A EP81108755 A EP 81108755A EP 0054676 A2 EP0054676 A2 EP 0054676A2
Authority
EP
European Patent Office
Prior art keywords
atp
hla
complement
lysed
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP81108755A
Other languages
German (de)
French (fr)
Other versions
EP0054676B1 (en
EP0054676A3 (en
Inventor
Reiner Dr. Scherer
Karl Dr. Wulff
Christine Treffert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Boehringer Mannheim GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics GmbH, Boehringer Mannheim GmbH filed Critical Roche Diagnostics GmbH
Priority to AT81108755T priority Critical patent/ATE5201T1/en
Publication of EP0054676A2 publication Critical patent/EP0054676A2/en
Publication of EP0054676A3 publication Critical patent/EP0054676A3/en
Application granted granted Critical
Publication of EP0054676B1 publication Critical patent/EP0054676B1/en
Expired legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing

Definitions

  • the invention relates to a method for determining HLA antigens on lymphocytes.
  • HLA antigens Human leukocyte antigens
  • HLA antigens are antigens of the histocompatibility system. They are responsible for the recognition of foreign tissue by the body and are therefore of great importance for organ transplants and blood transfusions, in which leukocytes are also transmitted.
  • HLA antigens are glycoproteins on the cell surface of nucleated cells and platelets. The easiest way to detect them is on lymphocytes. In addition to their importance as an intolerance factor in transplants, they also influence the course of life-determining biological processes, such as immunological recognition and immune response, as well as susceptibility to diseases.
  • the first and third antigen groups can be detected serologically with the help of specific antisera.
  • the HLA-D antigens cannot be determined serologically, but they are so strongly associated with the serologically defined HLA-DR antigens that an identity cannot be excluded. Around 80 different HLA features are currently serologically characterized.
  • lymphocytes It is known to HLA antigens on lymphocytes using the cytotoxicity test according to PI Terasaki and JD McClelland (Nature 2 0 4, 998-looo (1964)) and S. Kissmeyer-Nielsen and KE Kjerbye (Histocompatibility Test. 1967, 381-383 , ES Curtoni, PL Mattiuz, RM Posi, Ed.Munks Gaard, Copenhagen 1967). Lymphocytes obtained from venous blood by Ficoll-Isopaque gradient centrifugation are used as test cells. The lymphocytes are incubated with human antisera directed against certain HLA antigens and complement.
  • Complement is a serum component and consists of a number of protein factors that can activate each other in the form of an enzyme cascade (comparable to the blood coagulation system).
  • the first complement factor is activated by binding to an antigen-antibody complex. If the antigen is a cell, in this case a lymphocyte, the last step of the complement reaction leads to the lysis of the cell.
  • Human antisera specific for certain HLA antigens are obtained from pregnant women who usually produce antibodies against their children's HLA traits inherited from the father.
  • an indicator dye such as. B. Eosin
  • the object of the invention is now to provide a method for HLA determination that is simple to handle and that can be automated and that permits a more objective and reliable statement than the microscopic determination.
  • This object is achieved according to the invention by a method for determining HLA antigens on lymphocytes by incubating the lymphocytes with HLA antiserum and complement with the liberation of ATP from the cells lysed by complement, which is characterized in that the released ATP is characterized with an ATPase implemented, then the cells not lysed by complement quickly lysed and measured the ATP released therefrom.
  • lymphocyte suspension suitable for the test can be carried out by the customary methods, such as, for. B. by Ficoll-Isopaque gradient centrifugation.
  • the incubation of the lymphocytes with antiserum and complement is carried out in a known manner according to the cytotoxicity test method ( T erasaki, Kissmeyer-Nielsen) in a suitable buffer solution.
  • Suitable buffers are Hanks' buffered saline (Proc. Exp. Biol. Med. 71, 196-2oo) or D ulbeccomedium (J. Expt. Med. 99, 167- 182), pH 6.8 to 7.4.
  • Rabbit complement is preferably used as the complement.
  • ATP-degrading kinases can be used in the presence of a phosphate acceptor, e.g. B. Adenosine triphosphatase EC 3.6.1.3 or Apyrase EC 3.6.1.5 or ATP pyrophosphatase EC 3.6.1.8.
  • the enzyme Apyrase EC is preferred because of its great stability.
  • the z. B. can be obtained from potatoes, and catalyzes the reaction ATP + H 2 0 ⁇ ADP + P.
  • Apyrase is added in a concentration of 5 to 2ooo U / ml of the lymphocyte suspension.
  • Nonionic detergents such as. B. polyethylene oxide esters of alkyl and aralkyl carboxylic acids or corresponding ether. Suitable concentrations of detergent are 0.05 to 2%.
  • the released ATP is through a bioluminescence system, be standing determined from luciferin and luciferase. The determination is based on the following principle:
  • the amount of light released is proportional to the amount of ATP converted and can be measured in a predetermined time interval with a photometer.
  • the luminescence test is highly sensitive and detects less than 1 / ug ATP / 1 test solution.
  • the ATP determination is carried out in buffered solution. All buffers which buffer in a pH range between 5 and 9, preferably between 6 and 8, are suitable. Buffers which are able to maintain the constancy of the pH value with the lowest possible ionic strength are particularly preferred, such as e.g. B. Tris buffer, Hepes buffer and other good buffers.
  • the ATP determination system is added to the lymphocyte suspension at the same time as the detergent which causes the rapid release of the ATP.
  • Another object of the invention is a reagent for determining the ATP of the cells not lysed by complement, which is characterized in that it is a nonionic detergent, luciferin, luciferase, magnesium, complexing agent, apyrase, serum albumin and Contains buffer.
  • the Reaaenzkombination can in addition to the listed mandatory components in addition conventional solvents and auxiliaries, such as. B. stabilizers included.
  • An embodiment is particularly preferred which contains 0.1 to 1% detergent, 2-4 mg / l luciferase, 40 to 500 / umol / l D-luciferin, 4 to 12.5 mmol / l magnesium chloride, 0.1 to 1 Contains 3 mmol / 1 EDTA, 1 to 6 g / 1 bovine serum albumin, 10 to 100 U / l apyrase, 10 to 50 mmol / 1 buffer.
  • apyrase in the reagent according to the invention is necessary in order to suppress the background signal which is caused by the reagent alone. Without the presence of apyrase, an ATP determination up to a final concentration of about 10 -11 mol / 1 ATP is possible. The presence of apyrase in the reagent increases the sensitivity of the determination to 10 -13 mol / 1 ATP.
  • the apyrase in the reagent also degrades the ATP to be determined during the bioluminescence measurement, the measurement must be carried out at exact time intervals in order to ensure the reproducibility of the measurement.
  • the light measurement is started no later than 10 seconds after addition of the reagent according to the invention.
  • a high measurement signal indicates that under the reaction conditions no complement-dependent cell lysis occurred and therefore no antigen corresponding to the antiserum used was present on the cells.
  • a low measurement signal shows that an antigen-antibody-induced complement-related lysis has occurred, ie that a positive reaction has taken place in the system used.
  • a positive and a negative control are also determined.
  • the positive control corresponds to 100 %
  • the negative control corresponds to o% lysis.
  • the evaluation of the sample is based on the extent of the lysis: a lysis ⁇ 50% corresponds to a positive reaction, a lysis ⁇ 50% corresponds to a cross reaction and a lysis ⁇ 20 to 30% is assessed negatively.
  • a cross reaction occurs when an antiserum reacts with more than one HLA antigen. For example, one often finds cross-reactions between HLA-A2 and HLA-A28 or between HLA-B7, HLA-B27 and HLA-BW22.
  • the table shows a comparison between the microscopic determination and the ATP determination in the bioluminescence test in five test subjects. The agreement is complete except for Bw6 in subject 4. There could be an error in the microscopic reading.
  • the following examples further illustrate the invention. All bioluminescence measurements were made with the BIOLUMAT LB 95oo from Laboratorium Prof. BERTHOLD, Wildbad.
  • the reagent is left overnight in the dark at room temperature so that the ATP contained in the reagents can be broken down by the apyrase. Since the apyrase in the reagent degrades the ATP to be determined very quickly during the bioluminescence measurement, the measurement must be carried out in exact time. It is e.g. O at the time given as to 400 ul reagent 100 .mu.l ATP-containing sample solution and integrated after exactly 4.5 seconds for 1 0 seconds over the light emission. In this way it is possible to reliably and reproducibly determine ATP concentrations up to 10 -13 mol / 1 final concentration.
  • Triton N-1o1 or Triton X-100 is added to the reagent in a final concentration of 0.1%.
  • the suitable plastic tubes for the BIOLUMAT are used. A small depression of 0 approximately 4 mm and a depth of 0.3 mm is drilled into these plastic tubes. Before use, the tubes are freed of any ATP traces by pretreatment with dilute hydrochloric acid.
  • the following are pipetted together into the drilled well: 5 / ul of the lymphocyte suspension to be tested (5oo cells / / ul), 5 / ul HLA antiserum.
  • the tube is closed with a stopper and allowed to incubate at room temperature for 30 minutes.
  • the tubes are closed again and the solution is mixed well with the whirl mix.
  • the mixture is left to stand at room temperature for 60 minutes.
  • 400 ⁇ l of detergent-containing ATP reagent are added and the measurement is carried out immediately afterwards.
  • the lymphocytes are lysed in an extent of 5o to 1 00%, and the light emerging from the cells ATP is completely degraded by apyrase. Ideally, only one background signal is obtained with the ATP reagent.
  • the lymphocytes remain intact in the course of the immune reaction. They are then spontaneously quantitatively lysed by the detergent contained in the luminescent reagent, the emerging ATP causes a light signal corresponding to its concentration.
  • test tubes are prepared as described in Example 2. Then 5 ⁇ l of antiserum are added to the well and lyophilized. The reaction path is set by the addition of 5 to lo / l of a lymphocyte suspension corresponding titer and further carried out as described in Example 2.
  • This variant offers the advantage that a larger number of tubes loaded with a specific antiserum can be prepared and stored beforehand.

Abstract

1. Process for the determination of HL antigens on lymphocytes by incubation of the lymphocytes with HLA antiserum and complement, with the liberation of ATP from the cells lysed by complement, characterised in that one reacts the liberated ATP with an ATPase, subsequently rapidly lyses the cells not lysed by complement and determines the ATP liberated therefrom.

Description

Die Erfindung betrifft ein Verfahren zur Bestimmung von HLA-Antigenen auf Lymphozyten.The invention relates to a method for determining HLA antigens on lymphocytes.

Humanleukozyten-Antigene (HLA-Antigene) sind Antigene des Histokompatibilitätssystems. Sie sind verantwortlich für die Erkennung fremden Gewebes durch den Körper und haben daher eine große Bedeutung für Organtransplantationen und Bluttransfusionen, bei denen Leukozyten mit übertragen werden. HLA-Antigene sind Glykoproteine auf der Zelloberfläche von kernhaltigen Zellen und Thrombozyten. Sie lassen sich am einfachsten auf Lymphozyten nachweisen. Neben ihrer Bedeutung als Unverträglichkeitsfaktor bei Transplantationen, nehmen sie auch Einfluß auf den Ablauf lebensbestimmender biologischer Prozesse, wie die immunologische Erkennung und Immunantwort, sowie Empfänglichkeit für Erkrankungen.Human leukocyte antigens (HLA antigens) are antigens of the histocompatibility system. They are responsible for the recognition of foreign tissue by the body and are therefore of great importance for organ transplants and blood transfusions, in which leukocytes are also transmitted. HLA antigens are glycoproteins on the cell surface of nucleated cells and platelets. The easiest way to detect them is on lymphocytes. In addition to their importance as an intolerance factor in transplants, they also influence the course of life-determining biological processes, such as immunological recognition and immune response, as well as susceptibility to diseases.

Man unterscheidet nach ihren Genarten drei Gruppen von HLA-Antigenen.

  • 1) Die serologisch definierten Antigene (HLA-A, HLA-B, HLA-C) ;
  • 2) die lymphozytär definierten Antigene (HLA-D) und 3) die B-zelldefinierten Antigene (HLA-DR).
A distinction is made between three groups of HLA antigens according to their gene types.
  • 1) The serologically defined antigens (HLA-A, HLA-B, HLA-C);
  • 2) the lymphocytic antigens (HLA-D) and 3) the B cell-defined antigens (HLA-DR).

Die erste und dritte Antigengruppe kann serologisch mit Hilfe spezifischer Antiseren nachgewiesen werden. Die HLA-D-Antigene sind nicht serologisch bestimmbar, jedoch sind sie so stark mit den serologisch definierten HLA-DR-Antigenen assoziiert, daß eine Identität nicht ausgeschlossen wird. Zur Zeit sind rund 8o verschiedene HLA-Merkmale serologisch charakterisiert.The first and third antigen groups can be detected serologically with the help of specific antisera. The HLA-D antigens cannot be determined serologically, but they are so strongly associated with the serologically defined HLA-DR antigens that an identity cannot be excluded. Around 80 different HLA features are currently serologically characterized.

Die serologische Bestimmung von HLA-Antigenen auf Lymphozyten hat eine große Bedeutung in der Medizin vor allem auf folgenden Gebieten erlangt:

  • 1) Transplantation Es wurde gefunden, daß die Überlebenszeit eines Transplantats von einem mit dem Empfänger verwandten Spender größer ist, wenn Spender und Empfänger identische HLA-Antigene besitzen, d. h. die Gewebeverträglichkeit hängt von der Ähnlichkeit des HLA-Systems von Spender und Empfänger ab. Für Organtransplantationen und auch für die Transfusion von Leukozyten, Thrombozyten und anderen Blutbestandteilen ist daher eine genaue Kenntnis der HLA-Antigene von Spender und Empfänger erforderlich.
  • 2) Diagnose genetisch determinierter Krankheiten Bestimmte HLA-Antigenmuster sind mit einer Reihe verschiedenartiger Erkrankungen assoziiert, z. B. mit immunpathologischen Erkrankungen, primär-chronischer Polyarthritis, Coeliakie, Myasthenia gravis, Reitersyndrom, chronischer Hepatitis und möglicherweise auch Leukämie. Durch Bestimmung der HLA-Antigene ist die Möglichkeit für eine Frühdiagnose gegeben, so daß eine gezielte Therapie bereits vor Erscheinen der Symptome angewandt werden kann.
  • 3) Vaterschaftsgutachten Aufgrund der Vielzahl der Merkmale und des dominanten Erbgangs ist das HLA-System sehr gut für die Paternitätsserologie geeignet. Durch die Berücksichtigung von 21 HLA-Merkmalen konnte die Ausschlußwahrscheinlichkeit für Nichtväter bei Vaterschaftsgutachten auf 98,5 % erhöht werden.
The serological determination of HLA antigens on lymphocytes has gained great importance in medicine, particularly in the following areas:
  • 1) Transplantation It was found that the survival time of a transplant from a donor related to the recipient is longer if the donor and recipient have identical HLA antigens, ie the tissue tolerance depends on the similarity of the HLA system of donor and recipient. For organ transplants and also for the transfusion of leukocytes, thrombocytes and other blood components, precise knowledge of the HLA antigens of the donor and recipient is required.
  • 2) Diagnosis of Genetically Determined Diseases Certain HLA antigen patterns are associated with a number of different diseases, e.g. B. with immunopathological diseases, primary chronic polyarthritis, coeliaca, myasthenia gravis, equestrian syndrome, chronic hepatitis and possibly also leukemia. By determining the HLA antigens, the possibility of an early diagnosis is given, so that targeted therapy can be used before the symptoms appear.
  • 3) Paternity report Due to the variety of characteristics and the dominant inheritance, the HLA system is very well suited for paternity serology. By considering 21 HLA characteristics, the exclusion probability for non-fathers in paternity reports was increased to 98.5%.

Es ist bekannt, HLA-Antigene auf Lymphozyten mit Hilfe des Zytotoxizitätstests nach P. I. Terasaki und J. D. McClelland (Nature 204, 998-looo (1964)) und S. Kissmeyer-Nielsen und K. E. Kjerbye (Histocompatibility Test. 1967, 381-383, E. S. Curtoni, P. L. Mattiuz, R. M. Posi, Ed. Munks Gaard, Kopenhagen 1967) zu bestimmen. Als Testzellen werden Lymphozyten verwendet, die durch Ficoll-Isopaque-Gradientenzentrifugation aus venösem Blut gewonnen werden. Die Lymphozyten werden mit menschlichen Antiseren, die gegen bestimmte HLA-Antigene gerichtet' sind, und Komplement inkubiert. Komplement ist eine Serumkomponente und besteht aus einer Reihe von Proteinfaktoren, die sich gegenseitig in Form einer Enzymkaskade (vergleichbar dem Blutgerinnungssystem) aktivieren können. Der erste Komplementfaktor wird durch Bindung an einen Antigen-Antikörper-Komplex aktiviert. Ist das Antigen eine Zelle, in diesem Fall ein Lymphozyt, so führt der letzte Schritt der Komplementreaktion zur Lyse der Zelle.It is known to HLA antigens on lymphocytes using the cytotoxicity test according to PI Terasaki and JD McClelland (Nature 2 0 4, 998-looo (1964)) and S. Kissmeyer-Nielsen and KE Kjerbye (Histocompatibility Test. 1967, 381-383 , ES Curtoni, PL Mattiuz, RM Posi, Ed.Munks Gaard, Copenhagen 1967). Lymphocytes obtained from venous blood by Ficoll-Isopaque gradient centrifugation are used as test cells. The lymphocytes are incubated with human antisera directed against certain HLA antigens and complement. Complement is a serum component and consists of a number of protein factors that can activate each other in the form of an enzyme cascade (comparable to the blood coagulation system). The first complement factor is activated by binding to an antigen-antibody complex. If the antigen is a cell, in this case a lymphocyte, the last step of the complement reaction leads to the lysis of the cell.

Die für bestimmte HLA-Antigene spezifischen menschlichen Antiseren werden von schwangeren Frauen gewonnen, die gewöhnlich Antikörper gegen die vom Vater vererbten HLA-Merkmale ihrer Kinder bilden.Human antisera specific for certain HLA antigens are obtained from pregnant women who usually produce antibodies against their children's HLA traits inherited from the father.

Die bei erfolgter Immunreaktion durch Komplement lysierten Lymphozyten werden von den nichtlysierten Lymphozyten durch Anfärben mit einem Indikatorfarbstoff, wie z. B. Eosin, unterschieden. Das Ergebnis wird durch Betrachtung der Zellen im Phasenkontrastmikroskop ausgewertet.The lymphocytes lysed when the immune reaction is complemented by the non-lysed lymphocytes ten by staining with an indicator dye, such as. B. Eosin, distinguished. The result is evaluated by examining the cells in the phase contrast microscope.

Diese Methode hat wesentliche Nachteile: Die mikroskopische Bewertung des Ergebnisses ist weitgehend subjektiv und stellt eine Belastung für den Experimentator dar. Hierin liegt eine große Fehlermöglichkeit. Ein weiterer Nachteil ist, daß das Ergebnis in der Regel erst am nächsten Tag ausgewertet werden kann. Außerdem ist der Test in dieser Form für eine Automatisierung nicht geeignet.This method has major disadvantages: The microscopic evaluation of the result is largely subjective and is a burden for the experimenter. This is a great possibility of error. Another disadvantage is that the result can usually only be evaluated the next day. In addition, the test in this form is not suitable for automation.

Aufgabe der Erfindung ist es nun, ein in der Handhabung einfaches, automatisierbares Verfahren zur HLA-Bestimmung zu schaffen, das eine objektivere und sicherere Aussage erlaubt als die mikroskopische Bestimmung.The object of the invention is now to provide a method for HLA determination that is simple to handle and that can be automated and that permits a more objective and reliable statement than the microscopic determination.

Gelöst wird diese Aufgabe erfindungsgemäß durch ein Verfahren zur Bestimmung von HLA-Antigenen auf Lymphozyten durch Inkubation der Lymphozyten mit HLA-Antiserum und Komplement unter Freisetzung von ATP aus den durch Komplement lysierten Zellen, welches dadurch gekennzeichnet ist, daß man das freigesetzte ATP mit einer ATPase umsetzt, anschließend die durch Komplement nicht lysierten Zellen rasch lysiert und das daraus freigesetzte ATP mißt.This object is achieved according to the invention by a method for determining HLA antigens on lymphocytes by incubating the lymphocytes with HLA antiserum and complement with the liberation of ATP from the cells lysed by complement, which is characterized in that the released ATP is characterized with an ATPase implemented, then the cells not lysed by complement quickly lysed and measured the ATP released therefrom.

Es ist bekannt, daß bei der Zytolyse durch Komplement die Zellmembran für Metabolite durchlässig wird, so daß unter anderem auch ATP in das umgebende Medium austritt. Jedoch wird das ausgetretene ATP durch lymphozyteneigene ATPasen weitgehend abgebaut. Es war daher nicht zu erwarten, daß sich ein Test auf Basis der Messung von ATP für eine HLA-Antigenbestimmung eignen würde.It is known that the cell membrane for metabolites is transmissive at the cytolysis by complement, so that A T P escapes into the surrounding medium, among other things. However, the leaked ATP is largely broken down by lymphocyte-specific ATPases. It was therefore not to be expected that a test based on the measurement of ATP would be suitable for HLA antigen determination.

überraschenderweise wurde jedoch gefunden, daß in dem erfindungsgemäßen Testsystem trotz Anwesenheit von ATPasen eine ATP-Bestimmung möglich ist.Surprisingly, however, it was found that an ATP determination is possible in the test system according to the invention despite the presence of ATPases.

Die Erfindung hat gegenüber der herkömmlichen mikroskopischen Methode folgende Vorteile:

  • 1) Das Resultat ist objektivierbar, da es auf der Bestimmung eines meßbaren Parameters (ATP) beruht und nicht der subjektiven Beurteilung des Experimentators unterliegt wie beim mikroskopischen Test.
  • 2) Da alle Reaktionsschritte nur das Dosieren von Flüssigkeiten, den Transport von Lösungen und Inkubationen enthalten, ist das erfindungsgemäße Verfahren gut für eine Automatisierung geeignet.
  • 3) Die Qualität der verwendeten Lymphozyten ist unkritisch. Auch ein Anteil toter Zellen, z. B. bei Leukämie, der bei der mikroskopischen Bestimmung zu erheblichen Fehlern führen würde, stört hier nicht.
The invention has the following advantages over the conventional microscopic method:
  • 1) The result can be objectified, since it is based on the determination of a measurable parameter (ATP) and is not subject to the subjective assessment of the experimenter as in the microscopic test.
  • 2) Since all reaction steps contain only the dosing of liquids, the transport of solutions and incubations, the method according to the invention is well suited for automation.
  • 3) The quality of the lymphocytes used is not critical. A proportion of dead cells, e.g. B. with leukemia, which would lead to significant errors in the microscopic determination, does not bother here.

Die Herstellung einer für den Test geeigneten Lymphozytensuspension kann nach den üblichen Methoden, wie z. B. durch Ficoll-Isopaque-Gradientenzentrifugation, erfolgen.The preparation of a lymphocyte suspension suitable for the test can be carried out by the customary methods, such as, for. B. by Ficoll-Isopaque gradient centrifugation.

Die Inkubation der Lymphozyten mit Antiserum und Komplement wird in bekannter Weise nach der Methode des Zytotoxizitätstests (Terasaki, Kissmeyer-Nielsen) in einer geeigneten Pufferlösung durchgeführt. Geeignete Puffer sind Hanks' gepufferte Salzlösung (Proc. Exp. Biol. Med. 71, 196-2oo) oder Dulbeccomedium (J. Expt. Med. 99, 167-182), pH 6,8 bis 7,4. Als Komplement wird bevorzugt Kaninchenkomplement verwendet.The incubation of the lymphocytes with antiserum and complement is carried out in a known manner according to the cytotoxicity test method ( T erasaki, Kissmeyer-Nielsen) in a suitable buffer solution. Suitable buffers are Hanks' buffered saline (Proc. Exp. Biol. Med. 71, 196-2oo) or D ulbeccomedium (J. Expt. Med. 99, 167- 182), pH 6.8 to 7.4. Rabbit complement is preferably used as the complement.

Zum Abbau des aus den durch Komplement lysierten Zellen freigesetzten ATP können alle bekannten ATP abbauenden Kinasen in Gegenwart eines Phosphatakzeptors verwendet werden, z. B. Adenosintriphosphatase EC 3.6.1.3 oder Apyrase EC 3.6.1.5 oder ATP-Pyrophosphatase EC 3.6.1.8. Bevorzugt wird aufgrund seiner großen Stabilität das Enzym Apyrase EC. 3.6.1.5, das z. B. aus Kartoffeln gewonnen werden kann, und die Reaktion ATP + H20 → ADP + P katalysiert. Apyrase wird in einer Konzentration von 5 bis 2ooo U/ml der Lymphozytensuspension zugesetzt.To break down the ATP released from the complement lysed cells, all known ATP-degrading kinases can be used in the presence of a phosphate acceptor, e.g. B. Adenosine triphosphatase EC 3.6.1.3 or Apyrase EC 3.6.1.5 or ATP pyrophosphatase EC 3.6.1.8. The enzyme Apyrase EC is preferred because of its great stability. 3.6.1.5, the z. B. can be obtained from potatoes, and catalyzes the reaction ATP + H 2 0 → ADP + P. Apyrase is added in a concentration of 5 to 2ooo U / ml of the lymphocyte suspension.

Zum Aufschluß der durch Komplement nicht lysierten Zellen eignen sich alle Methoden, die die Zellmembran rasch zerstören oder für Metabolite durchlässig machen. In einer bevorzugten Ausführungsform werden die Zellen mit Hilfe von Detergentien lysiert. Besonders bevorzugt werden nichtionische Detergentien, wie z. B. Polyäthylenoxidester von Alkyl- und Aralkylcarbonsäuren bzw. entsprechende Äther eingesetzt. Geeignete Konzentrationen an Detergens sind o,o5 bis 2 %.All methods which rapidly destroy the cell membrane or make it permeable to metabolites are suitable for disrupting the cells not lysed by complement. In a preferred embodiment, the cells are lysed using detergents. Nonionic detergents, such as. B. polyethylene oxide esters of alkyl and aralkyl carboxylic acids or corresponding ether. Suitable concentrations of detergent are 0.05 to 2%.

Die ATP-Bestimmung kann nach einem der bekannten Verfahren erfolgen (Bergmeyer "Methoden der enzymatischen Analyse", Bd. 2, S. 2147). Ein Beispiel dafür ist die Bestimmung mit 3-Phosphoglyceratkinase, der die folgenden Reaktionsgleichungen zugrunde liegen:

Figure imgb0001
Figure imgb0002

  • PGK = 3-Phosphoglycerat-kinase EC. 2.7.2.3
  • GAPDH = Glycerinaldehyd-3-phosphat-dehydrogenase EC. 1.2.1.12
The ATP can be determined by one of the known methods (Bergmeyer "Methods of Enzymatic Analysis", Vol. 2, p. 2147). An example of this is the determination with 3-phosphoglycerate kinase, which is based on the following reaction equations:
Figure imgb0001
Figure imgb0002
  • PGK = 3-phosphoglycerate kinase EC. 2.7.2.3
  • GAPDH = glyceraldehyde-3-phosphate dehydrogenase EC. 1.2.1.12

Eine weitere Möglichkeit ist die Bestimmung von ADP mit Hexokinase und Glucose-6-phosphat-dehydrogenase. Der Reaktionsablauf ist in den folgenden Gleichungen dargestellt:

Figure imgb0003
Figure imgb0004

  • HK = Hexokinase EC. 2.7.1.1
  • G6P-DH = Glucose-6-phosphat-dehydrogenase EC. 1.1.1.49
Another possibility is the determination of ADP with hexokinase and glucose-6-phosphate dehydrogenase. The course of the reaction is shown in the following equations:
Figure imgb0003
Figure imgb0004
  • HK = Hexokinase EC. 2.7.1.1
  • G6P-DH = glucose-6-phosphate dehydrogenase EC. 1.1.1.49

Eine weitere Möglichkeit ist die Bestimmung von ATP mit Formyltetrahydrofolat-Synthetase EC 6.3.4.3 nach folgenden Reaktionsgleichungen:

Figure imgb0005
Figure imgb0006
5,1o-Methenyltetrahydrofolsäure besitzt ein Adsorptionsmaximum bei 35o nm und kann photometrisch durch Ermittlung der Extinktionszunahme bei dieser Wellenlänge bestimmt werden.Another possibility is the determination of ATP with formyl tetrahydrofolate synthetase EC 6.3.4.3 according to the following reaction equations:
Figure imgb0005
Figure imgb0006
 5,1o-methenyltetrahydrofolic acid has an adsorption maximum at 35o nm and can be determined photometrically by determining the increase in extinction at this wavelength.

In einer bevorzugten Ausführungsform der Erfindung wird das freigesetzte ATP durch ein Biolumineszenzsystem, bestehend aus Luciferin und Luciferase bestimmt. Die Bestimmung beruht auf folgendem Prinzip:

Figure imgb0007
Figure imgb0008
 In a preferred embodiment of the invention, the released ATP is through a bioluminescence system, be standing determined from luciferin and luciferase. The determination is based on the following principle:
Figure imgb0007
Figure imgb0008

Die freigesetzte Lichtmenge ist der umgesetzten ATP-Menge proportional und kann in einem vorbestimmten Zeitintervall mit einem Photometer gemessen werden. Der Lumineszenztest ist hochempfindlich und erfaßt weniger als 1/ug ATP/1 Versuchslösung.The amount of light released is proportional to the amount of ATP converted and can be measured in a predetermined time interval with a photometer. The luminescence test is highly sensitive and detects less than 1 / ug ATP / 1 test solution.

Die ATP-Bestimmung wird in gepufferter Lösung durchgeführt. Geeignet sind alle Puffer, die in einem pH-Bereich zwischen 5 und 9, bevorzugt zwischen 6 und 8, puffern. Besonders bevorzugt werden Puffer, die die Konstanz des pH-Werts bei möglichst niedriger Ionenstärke aufrechtzuerhalten vermögen, wie z. B. Tris-Puffer, Hepes-Puffer und andere Goodpuffer.The ATP determination is carried out in buffered solution. All buffers which buffer in a pH range between 5 and 9, preferably between 6 and 8, are suitable. Buffers which are able to maintain the constancy of the pH value with the lowest possible ionic strength are particularly preferred, such as e.g. B. Tris buffer, Hepes buffer and other good buffers.

Damit das freigesetzte ATP quantitativ erfaßt werden kann, bevor ein nennenswerter Abbau durch ATPasen erfolgt ist, wird das ATP-Bestimmungssystem gleichzeitig mit dem Detergens, das die rasche Freisetzung des ATP bewirkt, der Lymphozytensuspension zugesetzt.In order that the released ATP can be quantitatively recorded before significant degradation by ATPases has occurred, the ATP determination system is added to the lymphocyte suspension at the same time as the detergent which causes the rapid release of the ATP.

Ein weiterer Gegenstand der Erfindung ist ein Reagens zur Bestimmung des ATP der durch Komplement nicht lysierten Zellen, welches dadurch gekennzeichnet ist, daß es ein nichtionisches Detergens, Luciferin, Luciferase, Magnesium, Komplexierungsmittel, Apyrase, Serumalbumin und Puffer enthält. Die Reaaenzkombination kann außer den aufgeführten obligaten Bestandteilen zusätzlich übliche Lösungsmittel und Hilfsmittel, wie z. B. Stabilisatoren, enthalten.Another object of the invention is a reagent for determining the ATP of the cells not lysed by complement, which is characterized in that it is a nonionic detergent, luciferin, luciferase, magnesium, complexing agent, apyrase, serum albumin and Contains buffer. The Reaaenzkombination can in addition to the listed mandatory components in addition conventional solvents and auxiliaries, such as. B. stabilizers included.

Gemäß einer bevorzugten Ausführungsform enthält dieses Reagens

  • o,o5 bis 2 % nichtionisches Detergens,
  • o,1 bis 1o mg/1 Luciferase,
  • 15 bis 1000/umol/l D-Luciferin,
  • 2,5 bis 25 mmol/1 Magnesiumchlorid,
  • o,o5 bis 12,5 mmol/1 EDTA
  • o,o5 bis 12,5 g/l Rinderserumalbumin,
  • 5 bis 2000 U/1 Apyrase,
  • 5 bis 100 mmol/1 Puffer, pH 6 bis 8.
According to a preferred embodiment, this reagent contains
  • o, o5 to 2% non-ionic detergent,
  • 0.1 to 10 mg / 1 luciferase,
  • 15 to 1000 / umol / l D-luciferin,
  • 2.5 to 25 mmol / 1 magnesium chloride,
  • o, o5 to 12.5 mmol / 1 EDTA
  • o, o5 to 12.5 g / l bovine serum albumin,
  • 5 to 2000 U / 1 apyrase,
  • 5 to 100 mmol / 1 buffer, pH 6 to 8.

Besonders bevorzugt wird eine Ausführungsform, die o,1 bis 1 % Detergens, 2 bis 4 mg/1 Luciferase, 4o bis 5oo /umol/l D-Luciferin, 4 bis 12,5 mmol/1 Magnesiumchlorid, o,1 bis 1,3 mmol/1 EDTA, 1 bis 6 g/1 Rinderserumalbumin, 1o bis 1oo U/l Apyrase, 1o bis 5o mmol/1 Puffer enthält.An embodiment is particularly preferred which contains 0.1 to 1% detergent, 2-4 mg / l luciferase, 40 to 500 / umol / l D-luciferin, 4 to 12.5 mmol / l magnesium chloride, 0.1 to 1 Contains 3 mmol / 1 EDTA, 1 to 6 g / 1 bovine serum albumin, 10 to 100 U / l apyrase, 10 to 50 mmol / 1 buffer.

Die Anwesenheit weiterer Apyrase im erfindungsgemäßen Reagens ist erforderlich, um das Untergrundsignal, das durch das Reagens allein verursacht wird, zu unterdrükken. Ohne die Anwesenheit von Apyrase ist eine ATP-Bestimmung bis zu einer Endkonzentration von etwa 10-11 mol/1 ATP möglich. Die Anwesenheit von Apyrase im Reagens erhöht die Empfindlichkeit der Bestimmung auf 10-13 mol/1 ATP.The presence of further apyrase in the reagent according to the invention is necessary in order to suppress the background signal which is caused by the reagent alone. Without the presence of apyrase, an ATP determination up to a final concentration of about 10 -11 mol / 1 ATP is possible. The presence of apyrase in the reagent increases the sensitivity of the determination to 10 -13 mol / 1 ATP.

Da die Apyrase im Reagens auch während der Biolumineszenzmessung das zu bestimmende ATP abbaut, muß die Messung in exakten Zeitintervallen erfolgen, um die Reproduzierbarkeit der Messung zu gewährleisten. In einer bevorzugten Ausführungsform der Erfindung wird mit der Lichtmessung spätestens 1o Sekunden nach Zusatz des erfindungsgemäßen Reagens begonnen.Since the apyrase in the reagent also degrades the ATP to be determined during the bioluminescence measurement, the measurement must be carried out at exact time intervals in order to ensure the reproducibility of the measurement. In a be preferred embodiment of the invention, the light measurement is started no later than 10 seconds after addition of the reagent according to the invention.

Ein hohes Meßsignal zeigt an, daß unter den Reaktionsbedingungen keine komplementabhängige Zelllyse auftrat und damit kein dem verwendeten Antiserum entsprechendes Antigen auf den Zellen vorhanden war. Ein niedriges Meßsignal zeigt, daß eine Antigen-Antikörper-induzierte Komplementbedingte Lyse auftrat, daß also in dem verwendeten System eine positive Reaktion stattfand.A high measurement signal indicates that under the reaction conditions no complement-dependent cell lysis occurred and therefore no antigen corresponding to the antiserum used was present on the cells. A low measurement signal shows that an antigen-antibody-induced complement-related lysis has occurred, ie that a positive reaction has taken place in the system used.

Um das Meßergebnis zu differenzieren, wird je eine Positiv- und eine Negativkontrolle mitbestimmt. Die Positivkontrolle entspricht 100 %, die Negativkontrolle o % Lyse. Die Bewertung der Probe erfolgt nach dem Ausmaß der Lyse: Eine Lyse ≥ 5o % entspricht einer positiven Reaktion, eine Lyse < 5o % entspricht einer Kreuzreaktion und eine Lyse < 2o bis 3o % wird negativ bewertet. Eine Kreuzreaktion erhält man, wenn ein Antiserum mit mehr als einem bestimmten HLA-Antigen reagiert. Z. B. findet man häufig Kreuzreaktionen zwischen HLA-A2 und HLA-A28 oder zwischen HLA-B7, HLA-B27 und HLA-BW22.In order to differentiate the measurement result, a positive and a negative control are also determined. The positive control corresponds to 100 %, the negative control corresponds to o% lysis. The evaluation of the sample is based on the extent of the lysis: a lysis ≥ 50% corresponds to a positive reaction, a lysis < 50% corresponds to a cross reaction and a lysis <20 to 30% is assessed negatively. A cross reaction occurs when an antiserum reacts with more than one HLA antigen. For example, one often finds cross-reactions between HLA-A2 and HLA-A28 or between HLA-B7, HLA-B27 and HLA-BW22.

Die Tabelle zeigt bei fünf Probanden einen Vergleich zwischen der mikroskopischen Bestimmung und der ATP-Bestimmung im Biolumineszenztest. Die Übereinstimmung ist bis auf Bw6 bei Proband 4 vollständig. Hier könnte ein Fehler bei der mikroskopischen Ablesung vorliegen.

Figure imgb0009
Figure imgb0010
 Die folgenden Beispiele erläutern die Erfindung weiter. Alle Biolumineszenzmessungen erfolgten mit dem BIOLUMAT LB 95oo der Firma Laboratorium Prof. BERTHOLD, Wildbad.The table shows a comparison between the microscopic determination and the ATP determination in the bioluminescence test in five test subjects. The agreement is complete except for Bw6 in subject 4. There could be an error in the microscopic reading.
Figure imgb0009
Figure imgb0010
 The following examples further illustrate the invention. All bioluminescence measurements were made with the BIOLUMAT LB 95oo from Laboratorium Prof. BERTHOLD, Wildbad.

B e i s p i e 1 1Example: 1 1

Herstellung eines Reagens zur Bestimmung von aus Lymphozyten freigesetztem ATPPreparation of a reagent for the determination of ATP released from lymphocytes

1ooo ml Reagens enthalten:

  • Hochgereinigte Luciferase aus Photinus pyralis 2,5 mg D-Luciferin 45o/umol
  • Magnesiumchlorid 6,25o mmol
  • EDTA o,625 mmol
  • Rinderserumalbumin 4 g
  • HEPES-Puffer 2o mmol, pH 7,75
  • Apyrase aus Kartoffeln 5o Einheiten.
1ooo ml reagent contains:
  • Highly purified luciferase from Photinus pyralis 2.5 mg D-luciferin 45o / umol
  • Magnesium chloride 6.25o mmol
  • EDTA o, 625 mmol
  • Beef serum albumin 4 g
  • HEPES buffer 20 mmol, pH 7.75
  • Apyrase from potatoes 5o units.

Das Reagens wird über Nacht im Dunkeln bei Raumtemperatur stehengelassen, damit das in den Reagenzien enthaltene ATP durch die Apyrase abgebaut werden kann. Da die Apyrase im Reagens während der Biolumineszenzmessung das zu bestimmende ATP sehr rasch abbaut, muß bei der Messung im exakten Zeittakt gearbeitet werden. Es wird z. B. zur Zeit o zu 400µl Reagens 100µl ATP enthaltender Probelösung gegeben und nach exakt 4,5 Sekunden für 10 Sekunden über die Lichtemission integriert. Auf diese Weise ist es möglich, verläßlich und reproduzierbar ATP-Konzentrationen bis 10-13 mol/1 Endkonzentration zu bestimmen.The reagent is left overnight in the dark at room temperature so that the ATP contained in the reagents can be broken down by the apyrase. Since the apyrase in the reagent degrades the ATP to be determined very quickly during the bioluminescence measurement, the measurement must be carried out in exact time. It is e.g. O at the time given as to 400 ul reagent 100 .mu.l ATP-containing sample solution and integrated after exactly 4.5 seconds for 1 0 seconds over the light emission. In this way it is possible to reliably and reproducibly determine ATP concentrations up to 10 -13 mol / 1 final concentration.

Dem Reagens wird kurz vor der im Beispiel 2 beschriebenen Messung Triton N-1o1 oder Triton X-100in einer Endkonzentration von o,1 % zugegeben.Shortly before the measurement described in Example 2, Triton N-1o1 or Triton X-100 is added to the reagent in a final concentration of 0.1%.

B e i s p i e l 2Example: 2 Test auf HLA-AntigeneTest for HLA antigens

Es werden für die Messung die für die den BIOLUMAT passenden Kunststoffröhrchen verwendet. In diese Kunststoffröhrchen wird eine kleine Vertiefung von 0 etwa 4 mm und einer Tiefe von o,3 mm hineingebohrt. Die Röhrchen werden vor der Verwendung durch Vorbehandlung mit verdünnter Salzsäure von etwa vorhandenen ATP-Spuren befreit.For the measurement, the suitable plastic tubes for the BIOLUMAT are used. A small depression of 0 approximately 4 mm and a depth of 0.3 mm is drilled into these plastic tubes. Before use, the tubes are freed of any ATP traces by pretreatment with dilute hydrochloric acid.

In die gebohrte Vertiefung werden zusammen pipettiert: 5/ul der zu testenden Lymphozytensuspension (5oo Zellen/ /ul), 5/ul HLA-Antiserum.The following are pipetted together into the drilled well: 5 / ul of the lymphocyte suspension to be tested (5oo cells / / ul), 5 / ul HLA antiserum.

Das Röhrchen wird mit einem Stopfen verschlossen und 3o Minuten bei Raumtemperatur inkubieren gelassen.The tube is closed with a stopper and allowed to incubate at room temperature for 30 minutes.

Dann werden zugegebenThen be added

25µl Kaninchenkomplement und
65µl einer Apyraselösung (0,15 mU/1 in 2o mmol/1 Tris-Acetatpuffer, pH 7,75)25µl rabbit complement and
65μl of a Apyraselösung (0, 15 mU / 1 in 2o mmol / 1 Tris-acetate buffer, pH 7.75)

Die Röhrchen werden wieder verschlossen und mit dem Whirlmix wird die Lösung gut vermischt. Man läßt 60 Minuten bei Raumtemperatur stehen. Dann werden 400µl detergenshaltiges ATP-Reagens zugegeben und die Messung unmittelbar darauf durchgeführt.The tubes are closed again and the solution is mixed well with the whirl mix. The mixture is left to stand at room temperature for 60 minutes. Then 400 µl of detergent-containing ATP reagent are added and the measurement is carried out immediately afterwards.

Bei einer positiven Reaktion des Antiserums werden die Lymphozyten in einem Ausmaß von 5o bis 100 % lysiert, und das aus den Zellen austretende ATP wird durch die Apyrase vollständig abgebaut. Mit dem ATP-Reagens wird hier im Idealfall nur ein Untergrundsignal erhalten.In a positive reaction of the antiserum, the lymphocytes are lysed in an extent of 5o to 1 00%, and the light emerging from the cells ATP is completely degraded by apyrase. Ideally, only one background signal is obtained with the ATP reagent.

Liegt eine negative Reaktion des Antiserums vor, bleiben die Lymphozyten im Laufe der Immunreaktion intakt. Sie werden dann durch das im Lumineszenzreagens enthaltene Detergens spontan quantitativ lysiert, das austretende ATP verursacht ein seiner Konzentration entsprechendes Lichtsignal.If the antiserum has a negative reaction, the lymphocytes remain intact in the course of the immune reaction. They are then spontaneously quantitatively lysed by the detergent contained in the luminescent reagent, the emerging ATP causes a light signal corresponding to its concentration.

Mit polyvalentem HLA-Antiserum wurde im Fall einer positiven Reaktion unter den hier beschriebenen Bedingungen beispielsweise ein Untergrundsignal von 15ooo Impulsen pro 1o Sekunden erhalten. Bei negativer Reaktion (negatives Vergleichsserum) wird hier ein Signal der Größe 150000 Impulse pro 1o Sekunden erhalten. Die Präzision dieser Signale ist mit einem vk von weniger als 10 % so gut, daß sie eine sichere Unterscheidung zwischen positiver und negativer Reaktion erlaubt.With a polyvalent HLA antiserum, for example, in the event of a positive reaction under the conditions described here, an underground signal of 150,000 pulses per 10 seconds was obtained. In the event of a negative reaction (negative comparison serum), a signal of 150,000 pulses per 10 seconds is obtained. The precision of these signals is with a CV of less than 1 0% so good that it allows a reliable distinction between positive and negative reaction.

B e i s p i e l 3Example 3 Herstellung von antiserumhaltigen TeströhrchenManufacture of test tubes containing antiserum

Die Teströhrchen werden wie in Beispiel 2 beschrieben vorbereitet. Dann werden in die Vertiefung jeweils 5µl Antiserum gegeben und lyophilisiert. Durch Zugabe von 5 bis lo/ul einer Lymphozytensuspension entsprechenden Titers wird die Reaktion in Gang gesetzt und wie in Beispiel 2 beschrieben weiter durchgeführt.The test tubes are prepared as described in Example 2. Then 5 µl of antiserum are added to the well and lyophilized. The reaction path is set by the addition of 5 to lo / l of a lymphocyte suspension corresponding titer and further carried out as described in Example 2. FIG.

Diese Variante bietet den Vorteil, daß eine größere Anzahl von mit einem bestimmten Antiserum beschickten Röhrchen vorher bereitgestellt und gelagert werden können.This variant offers the advantage that a larger number of tubes loaded with a specific antiserum can be prepared and stored beforehand.

Claims (7)

1. Verfahren zur Bestimmung von HLA-Antigenen auf Lymphozyten durch Inkubation der Lymphozyten mit HLA-Antiserum und Komplement unter Freisetzung von ATP aus den durch Komplement lysierten Zellen, dadurch gekennzeichnet , daß man das freigesetzte ATP mit einer ATPase umsetzt, anschließend die durch Komplement nicht lysierten Zellen rasch lysiert, und das daraus freigesetzte ATP bestimmt.1. A method for determining HLA antigens on lymphocytes by incubating the lymphocytes with HLA antiserum and complement with the liberation of ATP from the cells lysed by complement, characterized in that the ATP released is reacted with an ATPase, then the complement is not lysed cells quickly lysed, and the ATP released therefrom determined. 2. Verfahren nach Anspruch 1, dadurch gekennzeichnet , daß man als ATPase Apyrase verwendet.2. The method according to claim 1, characterized in that apyrase is used as the ATPase. 3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet , daß man die durch Komplement nicht lysierten Zellen mit einem Detergens lysiert.3. The method according to claim 1 or 2, characterized in that one lyses the cells not lysed by complement with a detergent. 4.. Verfahren nach Anspruch 1 bis 3, dadurch gekennzeichnet , daß man zur ATP-Bestimmung ein Luciferin/Luciferase-System zusetzt und die in einem vorbestimmten Zeitintervall emittierte Lichtmenge mißt.4 .. Method according to claim 1 to 3, characterized in that a luciferin / luciferase system is added to the ATP determination and the amount of light emitted in a predetermined time interval is measured. 5. Verfahren nach Anspruch 1 bis 4, dadurch gekennzeichnet , daß die Lichtmessung spätestens 1o Sekunden nach Zusatz des Luciferin/Luciferase-Systems beginnt.5. The method according to claim 1 to 4, characterized in that the light measurement at the latest 10 seconds after the addition of the luciferin / luciferase system begins. 6. Reagens zur Bestimmung des ATP der durch Komplement nicht lysierten Zellen, dadurch gekennzeichnet , daß es ein nichtionisches Detergens, Luciferin, Luciferase, Magnesium, Komplexierungsmittel, Apyrase, Serumalbumin und Puffer enthält.6. Reagent for determining the ATP of the cells not lysed by complement, characterized in that it contains a nonionic detergent, luciferin, luciferase, magnesium, complexing agent, apyrase, serum albumin and buffer. 7. Reagens nach Anspruch 6, dadurch gekennzeichnet, daß es o,o5 bis 2 % nichtionisches Detergens, o,1 bis 1o m/l Luciferase, 15 bis 1000 µmol/l D-Luciferin, 2,5 bis 25 mmol/1 Magnesiumchlorid, 0,05 bis 12,5 mmol/1 EDTA, o,o5 bis 12,5 g/ml Rinderserumalbumin, 5 bis 2000 U/1 Apyrase und 5 bis 100 mmol/1 Puffer, pH 6 bis 8, enthält. 7. Reagent according to claim 6, characterized in that it o, o5 to 2% non-ionic detergent, 0.1 to 10 m / l luciferase, 15 to 1000 µmol / l D-luciferin, 2.5 to 25 mmol / 1 magnesium chloride, 0.05 to 12.5 mmol / 1 EDTA, o, o5 to 12.5 g / ml bovine serum albumin, 5 to 2000 U / 1 apyrase and Contains 5 to 100 mmol / 1 buffer, pH 6 to 8.
EP81108755A 1980-12-18 1981-10-22 Process for determining hl antigens Expired EP0054676B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT81108755T ATE5201T1 (en) 1980-12-18 1981-10-22 PROCEDURE FOR DETERMINING HL ANTIGENS.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19803047860 DE3047860A1 (en) 1980-12-18 1980-12-18 METHOD FOR DETERMINING HLA ANTIGENS
DE3047860 1980-12-18

Publications (3)

Publication Number Publication Date
EP0054676A2 true EP0054676A2 (en) 1982-06-30
EP0054676A3 EP0054676A3 (en) 1982-08-04
EP0054676B1 EP0054676B1 (en) 1983-11-02

Family

ID=6119575

Family Applications (1)

Application Number Title Priority Date Filing Date
EP81108755A Expired EP0054676B1 (en) 1980-12-18 1981-10-22 Process for determining hl antigens

Country Status (4)

Country Link
EP (1) EP0054676B1 (en)
JP (1) JPS57131063A (en)
AT (1) ATE5201T1 (en)
DE (2) DE3047860A1 (en)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987006707A1 (en) * 1986-04-29 1987-11-05 Murex Corporation Bound assay components
EP0389380A1 (en) * 1989-03-24 1990-09-26 Elf Sanofi Testkit and method applicable to whole cells
EP0389381A1 (en) * 1989-03-24 1990-09-26 Sanofi Kit and method for enzymatic determinations applicable to whole cells
WO1994017202A1 (en) * 1993-01-21 1994-08-04 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Microbiological test method and reagents
GB2289945A (en) * 1993-01-21 1995-12-06 Secr Defence Microbiological test method and reagents
WO1996002665A1 (en) * 1994-07-13 1996-02-01 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Microbiological test method and reagents
US5503976A (en) * 1982-07-30 1996-04-02 Biomerieux Sa DNA sequences coding for the DR β-chain locus of the human lymphocyte antigen complex and diagnostic typing processes and products related thereto
WO1996040988A1 (en) * 1995-06-07 1996-12-19 Promega Corporation Quenching reagents and assays for enzyme-mediated luminescence
US5648232A (en) * 1993-01-21 1997-07-15 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Microbiological best method and reagents
US6210891B1 (en) * 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
CN1091804C (en) * 1994-07-13 2002-10-02 大不列颠及北爱尔兰联合王国国防大臣 Microbiological test method and reagents
CN1125880C (en) * 1994-04-05 2003-10-29 研究技术集成责任有限公司 Method for processing mycobacteria
US6818393B1 (en) 1982-07-30 2004-11-16 Biomirieux Sa DNA sequences coding for the DR beta-chain locus of the human lymphocyte antigen complex and polypeptides, diagnostic typing processes and products related thereto

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1330594A (en) * 1970-08-04 1973-09-19 Nasa Urinalysis
DE2823816A1 (en) * 1977-05-31 1978-12-14 Inst Nat Sante Rech Med DEVICE FOR OBTAINING AND PROCESSING ELECTROCARDIOGRAPHICAL DATA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1330594A (en) * 1970-08-04 1973-09-19 Nasa Urinalysis
DE2823816A1 (en) * 1977-05-31 1978-12-14 Inst Nat Sante Rech Med DEVICE FOR OBTAINING AND PROCESSING ELECTROCARDIOGRAPHICAL DATA

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Band 92, Nr. 21, 26. Mai 1980, seite 294, Nr. 177041q Columbus Ohio, U.S.A. B. DESCAMPS: "Determination of Intracellular Adenosine Triphosphate for Detecting Anti-HLA Antibody-Mediated Cytolysis. Introduction to a New Method for HLA Typing" & Transplantation 1980, 29(4), 295-301 *
CHEMICAL ABSTRACTS, Band 93, Nr. 7, 18. August 1980, seite 440, Nr. 65272e Columbus, Ohio, U.S.A. F.J. FEHRENBACH et al.: "Measurement of Bacterial Cytolysins with Highly Sensitive Kinetic Method" & Fems Microbiol. Lett. 1980, 7(4) 285-288 *
Nature, Band 204, 5. Dezember 1964, seiten 998-1000 PAUL I. TERASAKI et al.: "Microdroplet Assay of Human Serum Cytotoxins" *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6818393B1 (en) 1982-07-30 2004-11-16 Biomirieux Sa DNA sequences coding for the DR beta-chain locus of the human lymphocyte antigen complex and polypeptides, diagnostic typing processes and products related thereto
US5503976A (en) * 1982-07-30 1996-04-02 Biomerieux Sa DNA sequences coding for the DR β-chain locus of the human lymphocyte antigen complex and diagnostic typing processes and products related thereto
WO1987006707A1 (en) * 1986-04-29 1987-11-05 Murex Corporation Bound assay components
EP0389380A1 (en) * 1989-03-24 1990-09-26 Elf Sanofi Testkit and method applicable to whole cells
EP0389381A1 (en) * 1989-03-24 1990-09-26 Sanofi Kit and method for enzymatic determinations applicable to whole cells
FR2644894A1 (en) * 1989-03-24 1990-09-28 Sanofi Sa KIT AND ENZYMATIC DETERMINATION METHOD APPLICABLE TO ENTIRE CELLS
FR2644893A1 (en) * 1989-03-24 1990-09-28 Sanofi Sa KIT AND METHOD OF DOSING APPLICABLE TO ENTIRE CELLS
US5648232A (en) * 1993-01-21 1997-07-15 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Microbiological best method and reagents
WO1994017202A1 (en) * 1993-01-21 1994-08-04 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Microbiological test method and reagents
GB2289945A (en) * 1993-01-21 1995-12-06 Secr Defence Microbiological test method and reagents
GB2289945B (en) * 1993-01-21 1997-06-18 Secr Defence Detecting microorganisms by assaying for ATP produced by intracellular adenylate kinase
CN1125880C (en) * 1994-04-05 2003-10-29 研究技术集成责任有限公司 Method for processing mycobacteria
GB2303919A (en) * 1994-07-13 1997-03-05 Secr Defence Microbiological test method and reagents
GB2303919B (en) * 1994-07-13 1998-08-26 Secr Defence Assay of a microorganism by quantification of adenylate kinase involving magnesium ion and adenosine diphosphate
CN1091804C (en) * 1994-07-13 2002-10-02 大不列颠及北爱尔兰联合王国国防大臣 Microbiological test method and reagents
WO1996002665A1 (en) * 1994-07-13 1996-02-01 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Microbiological test method and reagents
US5744320A (en) * 1995-06-07 1998-04-28 Promega Corporation Quenching reagents and assays for enzyme-mediated luminescence
WO1996040988A1 (en) * 1995-06-07 1996-12-19 Promega Corporation Quenching reagents and assays for enzyme-mediated luminescence
US6210891B1 (en) * 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation

Also Published As

Publication number Publication date
EP0054676B1 (en) 1983-11-02
DE3047860A1 (en) 1982-07-15
ATE5201T1 (en) 1983-11-15
DE3161331D1 (en) 1983-12-08
EP0054676A3 (en) 1982-08-04
JPS57131063A (en) 1982-08-13
JPS6232421B2 (en) 1987-07-14

Similar Documents

Publication Publication Date Title
EP0054676B1 (en) Process for determining hl antigens
JP3359921B2 (en) Suspension medium for blood composition and method of using the same
JP2557837B2 (en) Hematology control fluid suspension for three leukocyte populations and method of making same
US5320964A (en) Hematology control composition including leukocyte analogs; and methods for their preparation and use
JP4549440B2 (en) Reference control blood and recipe
US7824874B2 (en) Method for measuring in vivo mutation frequency at an endogenous gene locus
JP3822625B2 (en) Hematology control products
PT88615B (en) EQUIPMENT AND METHOD OF IMMUNOMETRIC DOSAGE APPLICABLE TO COMPLETE CELLS
JPH0726955B2 (en) Method for preparing preserved whole blood sample
EP0566010A1 (en) Method and analysis kit for determining the number of bacterial germs in whey and its components
US3689633A (en) Preparation of test sample for immunological assay of pregnancy of mares
DE2711489A1 (en) PROCESS FOR THE PRODUCTION OF STABILIZED ERYTHROCYTES AND THEIR USE AS AN INDICATOR FOR CARRYING OUT ANTIGEN-ANTIBODY REACTIONS
US6444471B1 (en) Reticulocyte containing complete blood control
Glass et al. Spontaneous-mutation rates at specific loci in Drosophila males and females
Denault et al. Detectability of selected genetic markers in dried blood on aging
DE60116689T2 (en) Whole blood immunoassay
US3764556A (en) Alcoholic precipitation and 1000x centrifugation preparation of hypercholesterolemic and hypertriglyceridemic sera as lipid determination control
US20200326331A1 (en) Methods and compositions for determining the presence or absence of dna aberrations
DE2906732B1 (en) Method and reagent for the determination of peroxidase
KR102261148B1 (en) Production of Glutaraldehyde-treated red blood cells with weakened expression of ABO and RhD antigens for use as a quality control material in tube technique
DE2636616C2 (en)
KR102609257B1 (en) An apparatus for testing abo antibody and method thereof
Muller et al. Detection of Immune Anti-A and Anti-B Alio-Antibodies with Groupamatic® System
Celano et al. Studies on eluates from rhesus and human A0 red cells
Kahl et al. Statistical Analysis of Monocyte Monolayer Assay Validation in Brazilian Blood Donors

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

PUAL Search report despatched

Free format text: ORIGINAL CODE: 0009013

17P Request for examination filed

Effective date: 19811022

AK Designated contracting states

Designated state(s): AT BE CH DE FR GB IT LU NL SE

AK Designated contracting states

Designated state(s): AT BE CH DE FR GB IT LU NL SE

ITF It: translation for a ep patent filed

Owner name: ING. C. GREGORJ S.P.A.

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

REF Corresponds to:

Ref document number: 5201

Country of ref document: AT

Date of ref document: 19831115

Kind code of ref document: T

REF Corresponds to:

Ref document number: 3161331

Country of ref document: DE

Date of ref document: 19831208

ET Fr: translation filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 19841026

Year of fee payment: 4

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 19841029

Year of fee payment: 4

26N No opposition filed
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 19841031

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 19841231

Year of fee payment: 4

Ref country code: BE

Payment date: 19841231

Year of fee payment: 4

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 19861021

Year of fee payment: 6

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 19871031

Year of fee payment: 7

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 19881021

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Effective date: 19891022

Ref country code: AT

Effective date: 19891022

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Effective date: 19891023

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Effective date: 19891031

Ref country code: CH

Effective date: 19891031

Ref country code: BE

Effective date: 19891031

BERE Be: lapsed

Owner name: BOEHRINGER MANNHEIM G.M.B.H.

Effective date: 19891031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Effective date: 19900501

NLV4 Nl: lapsed or anulled due to non-payment of the annual fee
GBPC Gb: european patent ceased through non-payment of renewal fee
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Effective date: 19900629

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Effective date: 19900703

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

EUG Se: european patent has lapsed

Ref document number: 81108755.0

Effective date: 19900705